Antigenic composition of a Pseudomonas aeruginosa protein

ABSTRACT

A novel antigen from  P. aeruginosa  is provided. The use of the antigen in detecting/diagnosing  P. aeruginosa  as well as its use in eliciting an immune response are also provided.

The present invention relates to a novel antigen from Pseudomonasaeruginosa, its use in medicine, particularly in the preparation ofvaccines and in diagnosis.

P. aeruginosa is a Gram-negative aerobic motile bacterium with the formof rods. It is an environmentally ubiquitous, extracellular,opportunistic pathogen that causes significant morbidity and mortalityin compromised subjects. Infection is of particular significance insubjects with cystic fibrosis, burns, chronic bronchitis, bronchiectasisand cancer.

Identification of immune responses, the search for vaccine candidatesand suitable components for diagnostic tests have focused on componentsof P. aeruginosa. The outer membrane of P. aeruginosa contains toxins,including the lipopolysaccharide endotoxin, phospholipid and proteins.The various outer membrane proteins (Opr) of P. aeruginosa have beenassigned an alphabetical naming system. While several proteins have beencharacterised by this scheme, the expression of some is only transientand highly dependent upon nutrient availability, culture conditions andthe presence of antibiotics. Presently, three major Oprs, designatedF,H2 and I, are recognised as antigenically common to and expressed inhigh copy numbers in all strains of P. aeruginosa.

We have now identified a protein from an outer membrane preparation ofP. aeruginosa, which we have designated Pa60. The amino-terminalsequence of this protein does not demonstrate any sequence homology withother previously characterised proteins (GenBank data search). Thisprotein is antigenic and is capable of inducing a protective immuneresponse resulting in enhanced clearance of P. aeruginosa.

Thus, in a first aspect the present invention provides a protein antigenfrom P. aeruginosa and having a molecular weight in the range of about60 kDa to about 65 kDa, as determined by SDS-PAGE.

In a preferred embodiment the protein has the following N-terminalsequence:

-   -   ?-E-E-K-?-?-L-?-?-?-?-?-?-?-V-V-?-N-A; and        preferably:    -   ?-E-E-K-T-P-L-T-T-A-A-?-A-P-V-V-?-N-A.

Parts or fragments of the whole protein may themselves be antigenic andthus, in a second aspect, the present invention provides an antigenicfragment of the protein of the invention. In particular, the antigenicfragment will comprise the N-terminal sequence as described above.

The skilled man will appreciate that some variation in the sequence offragments will be possible, while still retaining antigenic properties.Methods well known to the skilled man can be used to test fragmentsand/or variants thereof for antigenicity. Such variants also form partof the invention.

The antigenic protein, or fragments thereof, of the present inventioncan be provided alone, as a purified or isolated preparation, or as partof a mixture with other P. aeruginosa antigenic proteins.

In a third aspect, therefore, the invention provides an antigencomposition comprising one or more proteins of the invention and/or oneor more antigenic fragments thereof. Such a composition can be used forthe detection and/or diagnosis of P. aeruginosa. In one embodiment thecomposition comprises one or more additional P. aeruginosa antigens.

In a fourth aspect, the present invention provides a method of detectingand/or diagnosing P. aeruginosa which comprises:

-   -   (a) bringing into contact an antigenic protein, or antigenic        fragment thereof, or an antigen composition of the invention        with a sample to be tested; and    -   (b) detecting the presence of antibodies to P. aeruginosa.

In particular, the proteins, antigenic fragment thereof or antigencomposition of the invention can be used to detect IgG antibodies.Suitably, the sample to be tested will be a biological sample, e.g. asample of blood or saliva.

In a fifth aspect, the invention provides the use of an antigenicprotein, antigenic fragment thereof or antigenic composition of thepresent invention in detecting and/or diagnosing P. aeruginosa.Preferably, the detecting and/or diagnosing is carried out in vitro.

The antigenic protein, antigenic fragment thereof or antigen compositionof the invention can be provided as part of a kit for use in in vitrodetection and/or diagnosis of P. aeruginosa. Thus, in a sixth aspect,the present invention provides a kit for use in the detection and/ordiagnosis of P. aeruginosa comprising an antigenic protein, antigenicfragment thereof or antigen composition of the invention.

In addition, the antigenic protein or antigenic fragment thereof of theinvention can be used to induce an immune response against P.aeruginosa. Thus, in a further aspect, the present invention providesthe use of an antigen of the invention, a fragment thereof or anantigenic composition of the invention in medicine.

In yet a further aspect the present invention provides a compositioncapable of eliciting an immune response in a subject which comprises aprotein or one or more antigenic fragments thereof of the invention.Suitably, the composition will be a vaccine composition, optionallycomprising one or other suitable adjuvants. Such a vaccine compositionmay be either a prophylactic or therapeutic vaccine composition.

The vaccine compositions of the invention can include one or moreadjuvants. Examples of adjuvants well known in the art include inorganicgels such as aluminium hydroxide or water-in-oil emulsions such asincomplete Freund's adjuvant. Other useful adjuvants will be well knownto the skilled man.

In yet further aspects, the present invention provides:

-   -   (a) the use of a protein or one or more antigenic fragments        thereof of the invention in the preparation of an immunogenic        composition, preferably a vaccine;    -   (b) the use of such an immunogenic composition in inducing an        immune response in a subject; and    -   (c) a method for the treatment or prophylaxis of P. aeruginosa        infection in a subject, which comprises the step of        administering to the subject an effective amount of a protein,        at least one antigenic fragment or an antigen composition of the        invention, preferably as a vaccine.

Preferred features of each aspect of the invention are as for each otheraspect mutatis mutandis.

The invention will now be described with reference to the followingexample which should not be construed as limiting the invention in anyway.

The examples refer to the figure in which:

FIG. 1: shows SDS-PAGE analysis of Pa60.

EXAMPLE 1 Protein Purification

Pseudomonas aeruginosa bacteria, strain 385 (Pa385), were harvested fromovernight culture of 100 agar plates by scraping the plates followed bywashing twice by centrifugation at 10,000×g for 10 min at 4° C. A crudeouter membrane preparation was obtained by extraction of the outermembrane component with buffered Zwittergent 3-14 detergent and ethanolprecipitation.

The outer membrane extract was lyophilised and resuspended in startingbuffer (20 mM Tris, pH8.5). This preparation was subjected to anionexchange chromatography using a Q2 column (BioRad) and a sodium chloridegradient to elute the proteins. The fractions eluted from the columnwere initially assessed for protein content by analytical SDS-PAGE. Fromthis was determined the elution of profile for Pa60 allowing fractionscontaining Pa60 to be collected from susequent runs for furtherpurification. These fractions were dialysed against distilled water,lyophilised, resuspended in a minimal amount of distilled water andfurther dissolved in 4 times the volume of sodium dodecyl sulphate (SDS)reducing buffer (62.5 mM Tris, pH6.8, 10% (v/v) glycerol, 2% (w/v) SDS,5% (v/v) B-mercaptoethanol, 1.2×10⁻³% (w/v) bromophenol blue). The SDSpreparation was incubated at 37° C. for at least 30 min prior to beingloaded onto the stacking gel of the electrophoresis column.

Pa60 was purified using preparative polyacrylamide electrophoresis(PAGE). Preparative SDS-PAGE was performed using the BioRad model 491Prep Cell using a 9% T-1.42% C acrylamide/BIS(N,N′-methylene-bisacrylamide) separating gel with a 10 ml 4% T-0.36% Cacrylamide/BIS stacking gel polymerised in a 28 mm (internal diameter)column. Fractions eluted from the column were concentrated bylyophilisation and analysed for protein content by analytical SDS-PAGE.Pa60 isolated using these conditions contained SDS which wassubsequently removed by potassium phosphate precipitation. fractionscontaining Pa60 were pooled and dialysed prior to determination ofprotein concentration.

Analytical identification of the protein was performed by analyticalSDS-PAGE using either gradient 10-15% or homogenous 12.5% acrylamidegels and coomassie or silver stained. Protein concentration wasdetermined using the Pierce micro BCA assay.

Results

Pa60 was successfully separated from other P. aeruginosa proteins by thedescribed method. FIG. 1 shows the position of this protein on SDS-PAGE.

EXAMPLE 2 N-Terminal Sequencing of Pa60

Pa60 was prepared for N-terminal amino acid analysis by excising theregion containing the protein from an SDS-PAGE gel. The gel segmentswere sent to both the Biomolecular resouirce facility, AustralianNational University, Canberra, Australia and MUCAB Services, MacquarieUniversity, North ryde, NSW, Australia.

Results

An N-terminal amino acid sequence was obtained which identified sixteenof the first nineteen amino acids. Possible amino acids were identifiedfor the remaining residues and where there was uncertainty with aprobable identification. SEQUENCE: 1 2 3 4 5 6 7 8 9 10 Definite E E K LProbable T P T T A Possible S A L/S A I/D W 11 12 13 14 15 16 17 18 19Definite V V N A Probable A A P Possible F/L G/S N D

This provides a sequence with the following definite amino acids:1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19?-E-E-K-?-?-L-?-?-?-?-?-?-?-V-V-?-N-A

If one includes probable amino acids the following sequence is obtained:1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19?-E-E-K-T-P-L-T-T-A-A-?-A-P-V-V-?-N-A

EXAMPLE 3 Bacterial Clearance Following Immunisation in a Rat Model

Specific pathogen free male rats received an intra-Peyer's patch (IPP)immunisation on day 1 and the live bacterial challenge on day 14. Theanimals were sedated by anaesthesia. The small intestine was exposedthrough a mid-line abdominal incision and the antigen injectedsubserosal to each Peyer's patch using a 27-gauge needle. Theimmunisation protein (Pa60) was prepared by emulsification of 200 or 800μg of protein per ml in a 1:1 ratio of Incomplete Freund's adjuvant(IFA) and phosphate buffered saline (PBS) and a total inoculum of 10 or40 μg of protein respectively was administered to each animal. Animalswere challenged for 4 hours with live bacteria (bacteria count 5×10⁸CFU) 14 days after the immunisation. Bacteria were grown overnight at37° C. in 5% CO₂ on nutrient agar plates, recovered, washed andresuspended in PBS to the required concentration. Bacteria wereintroduced into the lungs via an intra-tracheal cannula and 4 hourslater the rats were euthanased. Blood was collected and aliquots ofserum stored at −20° C. for antibody analysis. Lungs were lavaged byflushing with 5×2 ml of PBS and the pooled lavage (BAL) assessed forbacteria numbers. Following lung lavage, the lungs were removed,homogenised and assessed for numbers of bacteria. Cytospin slides wereprepared for determination of differential cell counts in the lunglavage. total cell numbers present in the lung lavage were calculated bystaining with trypan blue and counting using a haemocytometer.

Results

Rats immunised with Pa60 and challenged with live bacteria of the Pa385homologous strain on day 14 showed an enhancement of bacterialclearance. Rats immunised with both 10 μg or 40 μg Pa60 had fewerbacteria recovered in both the BAL and lung than the non-immune groupafter 4 hours (Table 1).

Greater numbers of phagocytic cells were present in the BAL ofPa60-immunised animals and correlated with the enhanced bacterialclearance in these animals (table 2). TABLE 1 Pulmonary clearancefollowing Pa60 immunisation and challenge with P. aeruginosa (strain385) P. aeruginosa recovered 4 h post-challenge (log₁₀CFU)^(a) RAT GROUPn^(b) BAL LUNG HOMOG. NON-IMMUNE 5 7.63 ± 0.11 8.66 ± 0.18 10 μg Pa60 66.95 ± 0.07 8.43 ± 0.09 40 μg Pa60 4 7.19 ± 0.07 8.37 ± 0.19

TABLE 2 Cell count of Phagocytes in BAL following bacterial challengeTOTAL PHAGOCYTIC CELLS IN ANIMAL GROUP BAL NON-IMMUNE 1.2 (±0.3) × 10⁶10 μg Pa60 4.3 (±1.2) × 10⁶ 40 μg Pa60 7.4 (±1.7) × 10⁶

EXAMPLE 4 Clinical Diagnostic Study

Children from the Royal Children's Hospital in Melbourne that had beendiagnosed with cystic fibrosis provided samples for this study.Bronchoalveolar lavage (BAL) abnd serum were collected over a 3-4 yearperiod from patients from the time of diagnosis as an infant. Thesamples were divided into groups based on clinical status of P.aeruginosa.

-   -   Group 1: Non-cystic fibrosis controls (age matched children with        Stridor);    -   Group 2: Negative for P. aeruginosa;    -   Group 3: Upper respiratory tract isolation of P. aeruginosa,        negative P. aeruginosa in lower respiratory tract;    -   Group 4: Cleared P. aeruginosa in the lower respiratory tract        (negative in the next BAL sample); and    -   Group 5: Positive for P. aeruginosa in consecutive BAL samples.

An enzyme linked immunosorbent assay (ELISA) was used to measureantibodies to Pa60 in BAL and serum samples. Polysorb microtitre wellswere coated with purified Pa60 at a concentration of 1 g per ml. Theplates were washed five times in PBS containing 0.05% tween 20 betweenincubation steps. The wells were blocked with skim milk in PBS-0.05%Tween 20 for 60 min. Wells were incubated for 90 min with serum or BALsamples that were diluted in blocking buffer for analysis. Conjugatedimmunoglobulins used were rabbit anti-human IgG, IgA and IgM and wellswere incubated with conjugated immunoglobulins for 90 min. The plteswere then developed. Human IgG, IgA and IgM were used to quantitate theantibody.

Results

An increase in antibody titre was observed as the incidence of infectionwith P. aeruginosa occurred. The non-cystic fibrosis control group andthe non-infected cystic fibrosis patients had negligible titres to Pa60.Increased titres of IgG to Pa60 were observed, particularly in thepatients with consecutive P. aeruginosa culture from the BAL (Group 5).In the BAL a significant increase in IgA titre was observed. TABLE 3Pa60-specific Antibody in Serum and Bronchoalveolar lavage from cysticfibrosis and non-cystic fibrosis children SERUM^(a) BAL^(a) PATIENTS IgGIgA IgM IgG IgA IgM GROUP 1 1.74 0.11 0.67 0.03 0.05 0.02 GROUP 2 1.402.34 2.10 0.03 0   0.02 GROUP 3     7.08 ± 8.4    10.9 ± 18  2.03 ± 2.5 0.03 ± 0.01 0.21 ± 0.13 0.03 ± 0.01 GROUP 4    18.9 ± 21.9     0.56 ±0.6 1.46 ± 2.07 0.02 ± 0.01 0.12 ± 0.04 0.01 ± 0.01 GROUP 5    54.5 ± 76    7.5 ± 12.5 6.2 ± 0.5 0.03 ± 0.01 0.81 ± 0.30 0.03 ± 0.01

EXAMPLE 5

Pulmonary Challenge of rats with Pseudomonas aeruginosa followingmucosal immunisation with Pa60.

DA rats were immunised with Pa60 such that they received 10 μg Pa60administered to intestinal Peyer's Patches (IPP). The Pa60 was deliveredemulsified in Incomplete Freund's adjuvant. Fourteen days post-IPP, allthe immunised rats received an intra tracheal (IT) boost with 10 μg Pa60in phosphate buffered saline. Seven days post-IT boost, the immunisedgroup and an untreated control group were challenged via IT administeredof 5×10⁸ CFU live P. aeruginosa. The rats were killed and samplescollected for analysis at 4 h post-challenge.

Results Bacterial Clearance

Bacterial recovery in bronchoalveolar lavage (BAL) and lung tissueBacterial Recovery (log₁₀ CFU)* Group n+ BAL Lung Non-immune 5 7.92 ±0.11 9.01 ± 0.15 10 μg Pa60 5 6.34 ± 0.08 7.58 ± 0.08*Data expressed as mean ± S.E.M.+number of animals

Rats immunised IPP with an IT boost at day 14 significantly cleared theP. aeruginosa from both the BAL and lung tissue.

White Cell Recruitment to Infection White Cell Count in BAL

White cell Group n count* ×10⁶ non-immune 5  6.8 ± 1.4 10 μg Pa60 5 36.0± 5.0*data represents mean ± S.E.M.

Rats immunised with Pa60 more rapidly recruited white cells to the lungsfollowing bacterial challenge. Nearly all the white cells recovered inthe BAL were either polymorphonuclear neutrophils (PMNs) or macrophages.The early recruitment of white cells to the bronchoalveolar spacescorrelated with bacterial clearance.

Antibody Responses

Antibody in serum and BAL following mucosal immunisation with Pa60Antibody in Serum Group n IgG* IgA* IgM* non-immune 5 0 0 14.8 ± 0.56 10μg Pa60 5 1310 ± 119 10 ± 2 100 ± 59 *IgG, IgA and IgM expressed as Elisa units

Serum antibody to Pa60 was detected in immunised rats. There weresignificant titers of IgG, IgA and IgM.

Antibody in BAL

Group n IgG* IgA* IgM* non-immune 5  1.5 ± 0.2 0 0.5 ± 0.02 10 μg Pa60 521.3 ± 2.6 7.8 ± 4.2 0.8 ± 0.3 *IgG, IgA and IgM expressed as Elisa units

Antibody to Pa60 was detected in the BAL in immunised animals. Therewere significant titers of both IgG and IgA specific for Pa60.

In a particular embodiment, this invention provides a kit for detectionor diagnosis of P. aeruginosa in a sample from a patient. The kitcontains at least one or more antigens or antigenic fragments accordingto this invention, along with the means to detect binding between theantigens or fragments and antibodies which specifically bind suchantigens or fragments. Selection of suitable means for detectingantigen-antibody binding is easily within the skill of the ordinaryworker in this art, and include primary and/or secondary labeledantibodies to IgG from humans or other mammals, and/or other knownmaterials for sandwich assays, ELISA assays, competitive immunoassays,and other well known immunometric assay formats.

In yet another particular embodiment, this invention provides a methodfor diagnosing P. aeruginosa in a subject suffering from cysticfibrosis. This method comprises bringing into contact one of theproteins, antigenic fragments or antigen compositions disclosed in thisinvention with a biological sample obtained from a subject with cysticfibrosis. The biological sample is preferably a sample of mucous, e.g.saliva. This method further comprises detecting the presence ofantibodies to P. aeruginosa in such a sample by, for example, detectingbinding between the antigens or fragments and antibodies whichspecifically bind such antigens or fragments, using detection meanswhich are of common knowledge to those of skill in the art.

1-35. (canceled)
 36. A method for the treatment or prophylaxis of P.aeruginosa infection in a subject, which comprises the step ofadministering to the subject an effective amount of: i) an isolated P.aeruginosa protein having a molecular weight of about 60 kDa to about 65kDa and an N-terminal sequence comprising the amino acid sequence XaaGlu Glu Lys Thr Pro Leu Thr Thr Ala Ala Xaa Ala Pro Val Val Xaa Asn Ala(SEQ ID NO: 2), wherein Xaa is an unknown amino acid, or ii) an isolatedantigen fragment of a P. aeruginosa protein having a molecular weight ofabout 60 kDa to about 65 kDa, wherein said antigenic fragment comprisesat its N-terminus, the amino acid sequence: Xaa Glu Glu Lys Thr Pro LeuThr Thr Ala Ala Xaa Ala Pro Val Val Xaa Asn Ala (SEQ ID NO: 2), whereinXaa is an unknown amino acid, to elicit an immune response against P.aeruginosa in said subject.
 37. The method of claim 36, wherein thesubject is a human subject.
 38. The method of claim 37, wherein thesubject is suffering from cystic fibrosis.
 39. The method of claim 36,wherein an immunogenic composition comprising said protein or saidantigenic fragment is administered to the subject.
 40. The method ofclaim 37, wherein said immunogenic composition further comprises one ormore adjuvants.
 41. The method of claim 37, wherein said immunogeniccomposition further comprises a pharmaceutically acceptable carrier. 42.The method of claim 37, wherein said immunogenic composition furthercomprises one or more additional P. aeruginosa antigens.
 43. The methodof claim 37, wherein said immunogenic composition is a prophylacticvaccine composition.
 44. The method of claim 37, wherein saidimmunogenic composition is a therapeutic vaccine composition.